Drosophila RNP-4F is an ortholog to human p110, which functions with the pre-mRNA processing (PRP) protein class as a recycling factor to carry U4- and U6-snRNPs to the assembling spliceosome. Both inherited and acquired defects in pre-mRNA processing are being increasingly recognized as causes of human genetic diseases. A notable example of a relatively common (and also relevant to this proposal) human genetic disease which has been shown to be due to mutations in three different PRP genes (also present in Drosophila) is retinitis pigmentosa (RP), affecting 1 in 4000 individuals, being characterized by progressive retinal degeneration and eventually total blindness. The long-term objective of our research is to understand evolutionarily conserved cellular, developmental, molecular and genetic mechanisms behind regulation of genes which encode intron splicing assembly factor proteins. We are concentrating our efforts on posttranscriptional processes. Most translational control elements are located in the 5'- and 3'-UTR of eukaryotic mRNAs, and several depend on a cis-regulatory stem-loop secondary structure. Our preliminary studies show that the 5'-UTR region of one major Drosophila rnp-4f pre-mRNA isoform contains an evolutionarily-conserved primary and also secondary structure in which an intron pairs with the adjacent exon to form a long stable stem-loop. The region of interest undergoes alternative splicing to produce a second major mRNA isoform, and this process is developmentally regulated. We have proposed a negative feedback control model for modulation of RNP-4F translation, which may in turn itself be regulated by an alternative splicing decision. Our preliminary studies show that the Drosophila Dadar adenosine deaminase which acts on double-stranded RNA substrates in all eukaryotes may play a role in 5'-UTR alternative splicing regulation during embryogenesis. However, there are many different dADAR mRNA isoforms, and these have not been studied in embryos. We outline plans to elucidate the molecular mechanisms behind posttranscriptional expression control in the Drosophila splicing assembly factor RNP-4F and anticipate that the results may lead to discovery of corresponding processes in control of human p110. Specifically, we propose to: (1) Characterize the spatial and temporal expression of dADAR mRNA and protein isoforms during development; (2) Identify and evaluate the role of cis- and trans-acting factors in rnp-4f 5'-UTR intron splicing regulation;and (3) Evaluate the role of the rnp-4f 5'-UTR stem-loop structure in RNP-4F translational modulation during embryogenesis. We will accomplish these specific aims by: (a) using quantitative RT-PCR to identify and quantify specific dADAR mRNA isoforms during embryogenesis, accompanied by correlating the abundance peak of at least one isoform with that for rnp-4f long stem-loop mRNA;(b) using in situ hybridization to demonstrate correlation between the identified dADAR isoform and rnp-4f long stem-loop mRNA;(c) using developmental Westerns to show that the identified dADAR mRNA isoforms are translated into protein;(d) using RNA electrophoretic mobility shift assay to detect protein factors which directly bind the rnp-4f long stem-loop;(e) using MALDI-TOF mass spectrometry to identify the stem-loop binding protein factors;and (f) using transgenic GFP reporter gene fly lines to evaluate the role of the long stem-loop in rnp-4f translational modulation. This research will provide a basis for understanding the molecular mechanisms behind expression control in the Drosophila splicing assembly factor RNP-4F, and may provide a model for elucidation of expression control in the orthologous gene in other organisms, including humans. PUBLIC HEALTH RELEVANCE: The overall goal of this study is to understand the molecular mechanisms behind posttranscriptional control of the evolutionarily-conserved splicing assembly factor RNP-4F in the important genetic model organism Drosophila melanogaster. Specifically, we postulate that an isoform of dADAR adenosine deaminase plays a major role in a developmentally-regulated 5'-UTR alternative splicing decision, stabilizing a cis-acting evolutionarily-conserved stem-loop and subsequently enabling the modulation of RNP-4F translation by a negative feedback mechanism. In this proposal, we outline experiments which are designed to result in understanding the roles of cis- and trans-acting regulatory factors in genetic regulation of this evolutionarily-conserved splicing assembly factor.